Rotavirus reassortant vaccine

ABSTRACT

The present invention provides novel rotavirus reassortants, vaccines employing the novel reassortants and methods for their preparation and administration. One such reassortant contains the gene encoding the v.p.7 neutralization antigen of a human rotavirus. Another reassortant contains the gene encoding the v.p.4 neutralization antigen of a human rotavirus. The remaining genes are provided solely from the bovine rotavirus WC3 strain, or from both the human and bovine strains.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of pending U.S. patent application Ser. No.08/456,906, filed Jun. 1, 1995 and issued as U.S. Pat. No.5,750,109 onMay 12, 1998, which is a continuation of U.S. patent application Ser.No. 08/353,547, filed Dec. 9, 1994 and issued as U.S. Pat. No. 5,626,851on May 6, 1997, which is a continuation-in-part of U.S. patentapplication Ser. No. 08/121,220, filed Sep. 14, 1993 (now abandoned),which was a continuation of U.S. patent application Ser. No. 07/558,884,filed Jul. 26, 1990 (now abandoned), which is a continuation-in-part ofU.S. patent application Ser. No. 07/126,477, filed Nov. 30, 1987 (nowabandoned). Grandparent U.S. patent application Ser. No. 08/353,547identified above is also a continuation-in-part of U.S. patentapplication Ser. No. 08/249,696, filed May 26, 1994 (now abandoned),which is a continuation of U.S. patent application Ser. No. 07/902,321,filed Jun. 22, 1992 (now abandoned). These applications are incorporatedby reference herein.

FIELD OF THE INVENTION

The present invention refers generally to novel rotavirus reassortants,vaccines employing the novel reassortants and methods for theirpreparation and administration.

BACKGROUND OF THE INVENTION

Rotaviruses are the single most important agent of acutegastroenteritis, a disease which requires hospitalization of infants andyoung children in developed countries, and a frequent cause of death inchildren less than 5 years of age in developing regions of the world.Studies in the United States, Australia, and Japan have demonstratedthat between 34 and 63% of hospitalizations of children for acutediarrheal disease are associated with rotavirus infection [A. Z.Kapikian et al, Rev. Infect. Dis., 2:459-469 (1980)]. The incidence ofhospitalization for rotavirus gastroenteritis in a health maintenanceorganization in the U.S. was estimated to be 222 per 100,000 in childrenfrom 13 to 24 months of age, and 362 per 100,000 in those less than oneyear [W. Rodriguez et al, Am. J. Dis. Child., 134:777-779 (1980)].Infection with rotavirus was associated with 63% of all hospitalizationsfor acute diarrhea in this pediatric population [W. Rodriguez et al,cited above]. A recent review of mortality data in the U.S. from 1973 to1983 indicated that 500 deaths per year occur in children less than 4years old due to diarrheal diseases, and that 20 to 80% of excess winterdeaths due to diarrhea in the U.S. are associated with rotavirusinfections [M-S. Ho et al, JAMA, 260:3281-3285 (1988)]. Rotaviruses arealso responsible for a substantial proportion of the mortalityassociated with diarrheal diseases in third world countries. Aneffective rotavirus vaccine would therefore have a major impact on thehealth of children in both the developed and developing areas of theworld.

Rotaviruses have an inner and outer capsid with a double-stranded RNAgenome formed by eleven gene segments. Multiple serotypes have beendefined by plaque reduction neutralization tests, and studies ofreassortant viruses have demonstrated that two outer capsid proteins,v.p.7 and v.p.4, are the determinants of virus serotype. The v.p.7protein is coded for by either gene segment 7, gene segment 8 or genesegment 9 of the particular human rotavirus. The location of the v.p.7encoding gene may be determined for each specific rotavirus byconventional experimental methods. The protein v.p.4 is an 88,000 daltonmajor surface structural protein product of gene 4 of a rotavirus. Likev.p.7, it functions as a major serotype-specific antigen, operative inserum neutralization (SN) tests, capable of inducing serotype-specificneutralizing antibody, and capable in a mouse system of inducingserotype specific immune protection against rotavirus disease. [See,Offit et al, (1986) supra]. In some earlier references, the v.p.4 wasreferred to as v.p.3. After 1988, a change in nomenclature, resulted inthe more proper reference to this protein as v.p.4 [M. Liu et al,Virol., 163:26-32 (1988) and M. K. Estes et al, Immunol. Invest.,18:571-581 (1989)].

Since the gene segments encoding the v.p.7 and v.p.4 proteins segregateindependently [Y. Hoshino et al, Proc. Natl. Acad. Sci. USA,82:8701-8704 (1985) and P. Offit et al, J. Virol., 57:376-378 (1986)],it has been proposed that serotyping nomenclature include both the Gtype, determined by v.p.7, and the P type, determined by v.p.4 [M. Esteset al, Microbiological Reviews, 53:410-449 (1989)]. Most human rotavirusinfections in the U.S. are caused by viruses of G types 1, 2, 3, or 4,and P types 1, 2, or 3 [V. Gouvea et al, J. Infect. Dis., 162:362-367(1990), P. Woods et al, J. Clin. Microbiol., 30:781-785 (1992), and J.Gentsch et al, J. Clin. Microbiol., 30:1365-1373 (1992)]. However, otherhuman rotavirus types, including for example, type G9, are moreprevalent in Asia, Europe and certain third world countries.

A number of animal rotaviruses are attenuated in humans, and have beenevaluated as potential live rotavirus vaccines, including the bovineserotype G6 WC3 rotavirus. The WC3 vaccine virus was shown to beimmunogenic and non-reactinogenic in infants [H F. Clark et al, AmericanJournal Diseases of Children, 140:350-356 (1986) and H F. Clark et al,J. Infect. Dis., 158:570-587 (1988)], but was inconsistent in providingprotective immunity against human rotavirus infection [H F. Clark et al,J. Infect. Dis., 158:570-587 (1988), D. Bernstein et al, J. Infect.Dis., 162:1055-1062 (1990), and M. Georges-Courbot et al, Res. Virol.,142:405-411 (1991)]. However, it has been proposed thatserotype-specific immunity is necessary to induce consistent protectionagainst rotavirus diarrhea [J. Flores et al, Lancet, 1:882-884 (1987)and C. Christy et al, Pediatr. Infect. Dis., 7:645-650 (1988)].

There exists a need in the art for effective vaccines providingprotective immunity against rotavirus infection and the severe clinicalsymptoms associated therewith.

SUMMARY OF THE INVENTION

The present invention provides rotavirus reassortants useful as safe andefficacious vaccines for preventing human rotavirus infection and theclinical symptoms associated with severe rotavirus disease.

In one aspect the invention provides a rotavirus reassortant derivedfrom human and non-human rotavirus parental strains, which reassortantcontains from a human rotavirus parental strain at least the gene (orgene segment) encoding the virion surface protein v.p.7 neutralizationantigen. The non-human gene segments of the reassortant are preferablyprovided by the WC3 bovine rotavirus strain or progeny thereof.

In another aspect the invention provides a rotavirus reassortantcontaining from a human rotavirus parental strain at least the geneencoding the v.p.4 neutralization antigen. The non-human gene segmentsare preferably provided from the WC3 bovine rotavirus parental strain orprogeny thereof.

As another aspect of this invention there is provided a vaccine forproviding immunological protection against acute diarrhea caused byhuman rotavirus which contains at least one of the novel rotavirusreassortants of the present invention. One particularly preferredunivalent vaccine of the invention comprises the reassortant WI79-3,9(serotype G1). Particularly desirable vaccines contain the reassortantWI79-3,9 in combination with at least one other rotavirus reassortantselected from a G2, G3, G4, P1 and P2 rotavirus reassortant of theinvention.

Another aspect of the invention provides a method of vaccinating humansagainst human rotavirus infection employing the reassortant vaccines ofthe invention. This vaccination method may also employ more than one ofthe vaccine compositions of this invention.

Other aspects and advantages of the present invention will be apparentupon consideration of the following detailed description of theinvention, including illustrative examples of the practice thereof.

DETAILED DESCRIPTION OF THE INVENTION

This invention involves rotavirus reassortants suitable for use asvaccines, which are characterized by safety to humans and the ability toconfer immune protection against human rotavirus infection. Thereassortants are produced by genetic reassortment between an attenuatedbovine rotavirus (preferably WC3 or progeny thereof) and at least onerotavirus representing an epidemiologically important human serotype. Inone embodiment of this invention, the human rotavirus contributes to thereassortant at least the gene segment encoding the v.p.7 protein. Inanother embodiment of this invention, the human rotavirus parentcontributes to the reassortant at least the gene segment encoding thev.p.4 protein. In still a further embodiment, the human rotavirusparental strain contributes at least both the v.p.7 and v.p.4 genesegments. In additional embodiments, the human rotavirus parental strainmay contribute gene segments in addition to those which encode the v.p.7and/or v.p. 4 antigens.

The human rotavirus gene which encodes for the neutralization antigenv.p.7 and/or v.p.4 in the novel reassortant may be selected from anyhuman rotavirus serotype for which immunization is desired. Desirably,in a reassortant of this invention the v.p.7 gene is derived from a G1,G2, G3, or G4 human rotavirus serotype and the v.p.4 protein is derivedfrom a human P1 or P2 serotype. Among the rotavirus strains noted to beclinically significant in human rotavirus infections (hereinafter "humanrotavirus strains"), including strains useful in the present invention,are the strains provided below:

serotype G1: WI79, Wa, D;

serotype G2: strains WISC2 and DS1;

serotype G3: strains WI78, P, HCR3A;

serotype G4: Bricout (Br) B, ST3;

serotype G8: 69M;

serotype G9: WI61;

serotype P1: WI79, WI78, WI61, Wa;

serotype P2: DS1; and

serotype P3: WISC2; BrB; BrA, M37.

This list of human rotavirus strains is non-exclusive. For example,several rotavirus strains previously identified in animal infectionshave also been found in human infections. These strains may beanticipated to be useful as `human` rotavirus strains for the purposesof this invention, e.g., the `porcine` rotavirus OSU, a serotype G5, andthe `bovine` rotavirus B223, a serotype G10. One of skill in the art mayreadily obtain other appropriate human strains from suitabledepositaries or academic or commercial sources. Alternatively, othersuitable human rotavirus strains may be isolated and adapted to growthon a suitable cell line, e.g. MA104, Vero cells, or the like, usingknown techniques. See, e.g., Clark et al (1987) (describing isolation ofWI61 strain) and Example 1.

The non-human genes present in the reassortants of this invention areobtained preferably from the attenuated, serotype G6, bovine rotavirusstrain WC3 or its progeny, described in detail in U.S. Pat. No.4,636,385. The disclosures of that patent are incorporated by referenceherein to provide additional information about this rotavirus strain.WC3 replicates to a high titer in CV-1 cells (ATCC CCL70) and in Verocells (ATCC CCL-81) and is known to be attenuated and immunogenic inhuman infants [Clark et al, Amer. J. Dis. Children, 140:350 (1986)].

Representative isolates of this strain type which may be substituted forWC3 are WC2, WC4, WC5, WC6, WC7, WC8, WC9 and WC10. These bovinerotaviruses are readily distinguishable from other strains of bovinerotavirus by their distinctive RNA electropherotype, their failure tohemagglutinate primate red blood cells, their plaque morphology andresponse in the SN test.

Particularly desirable reassortants provided by the present inventioncontain the gene encoding the v.p.7 protein contributed by the selectedhuman rotavirus. The selected human rotavirus may also be attenuated, ifdesired, for use in the reassortant. The gene encoding the v.p.4 proteinmay be contributed by attenuated bovine rotavirus WC3 or by the humanrotavirus. The remaining gene segments are contributed by either thehuman or animal parental rotavirus, or both.

Throughout the specification, specific reassortants are designated byreference to the human strain which designates the gene or genomesegment (aka gene segment) encoding the v.p.7 or v.p.4 protein antigen.Following this human parental strain, e.g. WI79, the confirmed humangene segments present in the bovine WC3/human rotavirus reassortant areidentified by segment number. For example, reassortant WI79-3,9 containshuman gene segments 3 and 9 from strain WI79. Originally thisreassortant was designated WI79-9 to indicate that it contained the WI79v.p.7 encoding gene segment. The presence of human WI79 gene segment 3,which runs closely between the human and bovine strains on the gels, wasconfirmed via polymerase chain reaction (PCR). However, gene 3 from WI79was always present in this reassortant which was deposited on Nov. 25,1987 with the American Type Culture Collection (ATCC), 12301 ParklawnDrive, Rockville, Md. 20852 (U.S.A.). (See Table 1 below).

Alternatively, a rotavirus reassortant of this invention may beconstructed which contains genes from more than one parental humanrotavirus strain, as well as from the bovine rotavirus parental strain.One example of such a reassortant is WI79-3+WISC2-9, which contains genesegment 3 from WI79 and gene segment 9 (which encodes the v.p.7 antigen)from strain WISC2, as well as the bovine gene segments.

Table 1 below provides examples of particularly desirable reassortantscontaining the human v.p.7 gene segment and/or the human v.p. 4 genesegment. These reassortants are listed in association with their G or Pserotype in the order of their present desirability as representativesof that serotype. As indicated in Table 1, many of the reassortants andparental rotavirus strains were deposited with the American Type CultureCollection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852 (U.S.A.),an accepted Depositary Authority as indicated, on the dates specifiedabove and were given ATCC designations upon viability testing.

                  TABLE 1                                                         ______________________________________                                        Human     Parent or                                                           Serotype  Reassortant  ATCC #    Deposit Date                                 ______________________________________                                        G1        WI79-3,9.sup.a                                                                             VR2194    Nov. 25, 1987                                                       VR2196    Nov. 25, 1987                                          WI79-4,9     VR2415    July 8, 1993                                 G2        WI79-3 + WISC2-9       Dec. 7, 1994                                           WISC2                                                                         parental                                                                      strain       VR2417    July 8, 1993                                 G3        WI78-8                 Dec. 7, 1994                                           WI78-1,6-11  VR2195    Nov. 25, 1987                                          WI78-1,7-11.sup.b                                                   G4        Bricout B-9            Dec. 7, 1994                                 P1        WI79-4       VR2377    June 19, 1992                                          WI79-4,9     VR2415    July 8, 1993                                           WI61-4.sup.b                                                        P2        DS1-4.sup.b                                                         ______________________________________                                         .sup.a Originally named WI799. The two deposits represent different           passage levels of the reassortant.                                            .sup.b Not deposited.                                                    

The deposits of WI79-3,9 and WI78-1,6-11 have been converted to complywith the requirements of the Budapest Treaty. All other deposits havebeen originally made under the Budapest Treaty. All restrictions on theavailability to the public of the deposited material identified in Table1 will be irrevocably removed upon the grant of a patent on thisapplication, the culture(s) will be maintained for a period of 30 yearsfrom the deposit date, or at least five years after the most recentrequest for a sample, whichever is longer; and the deposit will bereplaced if viable samples cannot be dispensed by the depositary. Duringthe pendency of this patent application, access to these deposits willbe afforded to one determined by the Commissioner to be entitledthereto.

Production of Reassortants

The method for producing the novel reassortants of this inventionincludes the step of isolating the human and other species parentrotavirus by culturing in a suitable cell culture. Briefly, the parentviruses are used to co-infect a cell line by conventional techniques andthe progeny viruses are identified by running each parent and theprogeny on conventional gel electrophoresis. The parental viruses may beeither two rotavirus strains (e.g. Bricout B and WC3), or a rotavirusstrain and a selected reassortant (e.g. WISC2 and WI79-3,9). Each genesegment runs with a characteristic mobility. The makeup of thereassortant is readily identified by comparison of its electropherotypeprofile with that of the parent. Performing the infections and gelelectrophoresis techniques to obtain such reassortants are skills knownto the art. The isolation technique is standard and is described in moredetail in Example 1.

Suitable cells for such isolation and infection include primate VEROcells (ATCC CCL-81), African green monkey kidney cells CV-1 (ATCCCCL-70); BSC-1 (ATCC CCL-26). Preferably, fetal green monkey cellMA-104, and primary primate kidney cell cultures are utilized. Verocells are presently preferred for vaccine manufacture. For purposes ofthis invention, primary primate kidney cell cultures include first,second (secondary) or third (tertiary) passages of kidney cells derivedfrom the indicated species of primate. Each of these cell culturesubstrates may be grown in BHK medium [MacPherson, I and M. Stoker,Virology, 16:147 (1962)], supplemented with 10% fetal calf serum,Eagle's minimal essential medium with 10% fetal calf serum, or medium199 with 10% fetal calf serum. These media may also contain gentamicin,25 micrograms per milliliter. These cell lines may be used alone, or inalternate passages of the viruses. When used in combination, a separatebut different cell line can be used in each of the various passages ofthe virus.

For example, a suitable cell culture is infected with both theattenuated bovine rotavirus strain WC3 and the desired human serotyperotavirus. Alternatively, a suitable cell culture may be infected withthe desired human parental strain and a human/WC3 reassortant of theinvention. Mixed infections are designed to maximize the potential forreassortment by ensuring that large and equal concentrations of eachparent virus are replicating simultaneously. After infection andsufficient time and conditions for gene reassortment, reassortantprogeny clones are examined by random selection of plaques, e.g., byperforming a plaque assay of the virus yield from the mixed infection.The virus is propagated in individual plaques which are induced byinoculation of the yield of the mixed infection onto another cellculture monolayer.

Polyacrylamide gel electrophoresis with silver stain (PAGE-SS) accordingto the procedure of Dolan et al, J. clin. Microbiol., 21:753 (1985) isemployed to analyze each such virus population and compare itselectropherotype with that of each parental rotavirus. The identity ofgenes which run closely (e.g., in certain human and bovine strains, genesegment 3 runs very closely on electropherotype gels), may optionally beconfirmed via known polymerase chain reaction techniques [J. Gentsch etal, J. Clin. Microbiol., 30:1365-1373 (1992); V. Gouvea et al, J. Clin.Microbiol., 28:276-282 (1990)].

The proportion of rotavirus reassortants isolated may be enhanced byselecting plaques whose morphology differs from that of either parent.Alternatively, progeny clones may be selected from the virus yield ofthe mixed infection after treatment with hyperimmune antiserum to theserotype of the rotavirus which does not contribute the desired proteinantigen-encoding gene [See, e.g., the method of U.S. Pat. No.4,571,385], prior to performing the plaque analysis of the population.This method may be applied to any human or animal virus.

Progeny clones are examined by harvesting individual plaques, which arethen cultivated individually in cell culture and examined for their geneconstitution by PAGE-SS. Reassortant progeny clones are selected asvaccine candidates if their PAGE-SS reveals the presence of at least thegene coding for the surface antigen v.p.7 from the human rotavirusagainst which immune protection is being sought, or the gene coding forthe surface antigen v.p. 4 from the human rotavirus against which immuneprotection is being sought. Preferably, the PAGE-SS will reveal thepresence of human v.p.7 in the reassortant. Alternatively, wheredesired, the PAGE-SS will reveal the presence of the human v.p.4 in thereassortant, or both the v.p.4 and v.p.7 encoding gene segments from thehuman rotavirus parental strain. other human genes may also be found inthe reassortant.

Vaccine Compositions

Vaccines for providing immunological protection against acute diarrheacaused by human rotavirus infection may contain one or more of the novelreassortants of the present invention. Desirable vaccine compositionslisted in order of from presently most preferred to presently lesserpreferred compositions are summarized in Table 2 and discussed in detailbelow.

                  TABLE 2                                                         ______________________________________                                        Vaccine Compositions                                                                         Preferred Reassortants                                         ______________________________________                                        G1 + G2 + G3 + G4                                                                            WI79-3,9 + (WI79-3 + WISC2-9) +                                               WI78-8 + BrB-9                                                 G1 + G2 + G3 + G4 +                                                                          WI79-3,9 + (WI79-3 + WISC2-9) +                                P1             WI78-8 + BrB-9 + WI79-4                                        G1 + G2 + G3 + P1                                                                            WI79-3,9 + (WI79-3 + WISC2-9) +                                               WI78-8 + WI79-4                                                G1 + P1        WI79-3,9 + WI79-4                                              G1 + G2 + G3   WI79-3,9 + (WI79-3 + WISC2-9) +                                               WI78-8                                                         G1 + G2 + G3 + G4 +                                                                          WI79-3,9 + (WI79-3 + WISC2-9) +                                P1 + P2        WI78-8 + BrB-9 + WI79-4 +                                                     DS1-4                                                          G1             WI79-3,9                                                       ______________________________________                                    

One such vaccine, a univalent vaccine, contains a single G1 rotavirusreassortant. Preferably, this G1 reassortant is WI79-3,9 which has beenshown to be effective in preventing rotavirus gastroenteritis during asubsequent epidemic of predominantly serotype G1 rotavirus. Thisreassortant was also the subject of study provided in detail in Example5. Briefly, the study was prospective, randomized, double-blind,placebo-controlled study performed, conducted over a single rotavirusseason in a total of 325 infants who were aged 2-8 months at enrollment.The subjects were randomized to receive either placebo or WI79-3,9 virusvaccine at 10⁷.3 plaque forming units in three oral doses each separatedby 2 months and followed for seven days after each dose for occurrenceof adverse events and during the subsequent winter for development ofrotavirus gastroenteritis. It was found that administration of WI79-3,9virus vaccine was well tolerated, and rates of adverse events includingfever were similar in vaccine and placebo recipients. The live,attenuated serotype G1 bovine-human rotavirus reassortant WI79-3,9vaccine was found safe and effective in prevention of homotypic humanrotavirus infection in infants. See Example 5 below.

The vaccine compositions of the invention may desirably include otherrotavirus reassortants of the invention, in addition to a G1teassortant. Preferably, these reassortants are representative of one ormore of serotypes G2, G3, G4, P1, and P2, as listed above in Tables 1and 2. For example, in one desirable formulation, the invention providesa vaccine composition containing WI79-3,9 and WI79-4 (serotype G1 andP1, respectively). This composition has been shown to elicit a strongerimmune response than does a single reassortant rotavirus containing boththe human v.p.4 and the human v.p.7 (encoded by gene segment 9). SeeExample 6 below.

Other suitable combinations include a quadrivalent vaccine containing aG1 reassortant of the invention in combination with a G2, G3, and P1reassortant, e.g. WI79-3,9; WI79-3+WISC2-9; WI78-8; and WI79-4. SeeExample 7 below in which preliminary analysis of such a vaccine revealsgreater than 70% efficacy.

Suitable combination vaccines, which may be univalent, bivalent,trivalent, quadrivalent, quinquavalent or sexavalent may include variouscombinations of the G1, G2, G3, G4, P1 and P2 reassortants. Othersuitable reassortants of the invention may be selected for use invaccine compositions other than those specified in Table 2 by referenceto Table 1 above and the present specification.

The vaccines of the invention contain conventional carriers. Suitablecarriers are well known to those of skill in the art. These vaccinecompositions are preferably prepared in liquid unit dose forms. Otheroptional components, e.g. stabilizers, buffers, preservatives,excipients and the like, may be readily selected by one of skill in theart. However, the compositions may be lyophilized and reconstituted bythe attending physician prior to administration of the dose.Alternatively, the vaccine compositions may be prepared in any mannerappropriate for the chosen mode of administration, e.g. parenteral. Thepreparation of a pharmaceutically acceptable vaccine, having due regardto pH, isotonicity, stability and the like, is within the skill of theart.

When adapted for oral administration, one particularly desirableformulation includes as a carrier Williams E media/50% sucrose/0.1 Msuccinate/50 mM phosphate liquid. Another desirable formulation includes0.2M succinate and 0.1 M phosphate. When adapted for parenteraladministration, conventional adjuvants may also be employed in thevaccine composition, e.g., aluminum hydroxide.

Optionally, the vaccine may be formulated to contain other activeingredients and/or immunizing antigens. For example, when adapted fororal administration, formulation with the Sabin polio vaccine may bedesirable.

The dosage regimen involved in a method for vaccination, including thetiming, number and amounts of booster vaccines, will be determinedconsidering various hosts and environmental factors, e.g. the age of thepatient, time of administration and the geographical location andenvironment.

Method of Vaccination

Therefore, also included in the invention is a method of vaccinatinghumans against human rotavirus infection with the novel reassortants andvaccine compositions described above. The vaccine compositions includingone or more of the reassortants described herein are administered,preferably by the oral route, in a suitable dose, preferably liquid. Thevaccine may also be administered intranasally or parenterally.Alternatively, the vaccine may be administered to nursing mothers as ameans for transferring immunity to the infant.

The dosage for all routes of administration is generally greater than10⁶, between 10⁶ and 10⁹ plaque forming units (pfu) of the reassortant,with the preferred dosage being 10⁷ pfu. Additional doses of thevaccines may also be administered. It may be preferable to inoculatesusceptible infants and children on an annual basis prior to the"rotavirus season". Rotavirus infection in humans has been observed tooccur in various geographical regions during the same season, e.g. inwinter in the United States. Repeated inoculations prior to that seasonfor susceptible infants and children may be indicated. For example, onecurrently preferred dosage regimen for the U.S. includes three dosesapproximately two months apart prior to the beginning of the rotavirusseason.

The following examples illustrate the preferred methods for preparingthe rotavirus reassortant vaccines of the invention. These examples areillustrative only and do not limit the scope of the invention.

EXAMPLE 1 Isolation of the Rotaviruses

The bovine rotavirus strain WC3 and human rotavirus strains used inproducing reassortants according to the invention were isolated in cellline MA104 or in primary primate cells and then adapted to growth in theVero cell line.

The human origin rotaviruses were isolated by standard techniques asdescribed previously for isolation of human rotavirus strain WI61 inClark et al (1987) supra. Stools of infants ill with gastroenteritiswere determined to contain rotavirus by ELISA and/or by the PAGE-SStechnique for detection of the rotavirus-characteristic 11 segments ofdouble-stranded RNA. Rotavirus-containing stools were emulsified into a5% (w/v) suspension in serum-free Eagle's Minimal Essential Mediumcontaining 500 units of penicillin/ml, 500 micrograms ofstreptomycin/ml, 40 micrograms of gentamicin/ml 50 units of nystatin/ml,and 20 micrograms of trypsin/ml. The stool suspension was clarified bycentrifugation at 2000×g for 30 minutes.

Clarified supernatant fluid was incubated with an equal volume ofpurified trypsin (10 microgram/ml) in phosphate buffered saline (PBS)for 60 minutes at 37° C. The trypsin-treated stool supernatant fluid wasinoculated in a volume of 0.2 ml into tube cultures of MA104 cells whichhad previously been washed three times with PBS. After absorption ofthis rotavirus-containing fluid for 30 minutes at 37° C., the tubecultures were fed with 1.5 ml of Sato medium containing 1 microgram/mlof purified trypsin and incubated in a roller apparatus at 37° C.

Inoculated cell cultures were harvested after seven days of incubationby freezing and thawing of the combined cells and cell culture medium.Serial passage was accomplished by inoculating 0.2 ml of undiluted cellculture suspension into fresh tubes of MA104 cell culture treated in thesame manner as the initial passage inoculated with stool suspensionsupernatant fluid. Cell culture suspensions from each successive passagewere analyzed for the presence of rotavirus RNA by the PAGE-SStechnique. Detectable concentrations of rotavirus RNA were usuallyobtained by the second and third passage level. Visible cytopathiceffect (CPE) usually appeared by the second to fifth cell culturepassage.

After the rotavirus strain has become cytopathic, serial passages weremade whenever CPE involved more than 75% of the cell monolayer (2 to 7days). When a rotavirus isolate consistently induced CPE in roller tubecultures within 48 hours (usually within 4 to 8 passages), serialpassage was performed in stationary cultures of MA104 cells fed with BHKmedium supplemented with 13 micrograms/ml of unpurified trypsin (FlowLabs). Serial subculture in MA104 cell stationary cultures was performedin the same manner as that used for roller tubes, and was continueduntil the isolated rotavirus was determined to efficiently induceplaques under agarose overlay in MA104 cell culture.

When the rotavirus isolate efficiently induces plaques in the plaqueinduction assay according to Offit et al, J. Virol. Methods, 7:29 (1983)[usually 10⁵ to 10⁷ pfu per ml], it has adapted to growth in the MA104cell culture. It is then adapted to growth in stationary cultures ofCV-1 cells by similar serial passage methods, except that the medium isEagle's MEM serum-free, containing 6.25 microgram/ml unpurified trypsin(Flow). At varying passage levels, as appropriate, the isolatedrotavirus may be genetically purified by isolation and propagation of asingle plaque produced in MA104 cell culture. Mechanical aspiration ofcells within a single plaque, well separated from any surroundingplaques is followed by serial propagation of virus contained in thiscell suspension by standard technique.

It is presently preferred to inoculate the virus into tube cultures ofVero cells in the presence of serum-free medium with 0.75 μg/ml purifiedtrypsin. These cultures are incubated in roller apparatus at 37° C.,with the trypsin being replenished at 3 to 4 day intervals. The cultureis harvested by freezing and thawing about 7 to 10 days afterinoculation. Sub-passages are made in additional roller tubes of Verocells or stationary cultures of Vero cells in tissue culture flasks.After 2-5 passages, virus is capable of causing CPE in stationarycultures, and may be used to prepare the vaccines.

The identity of the cell culture-adapted rotavirus compared with thevirus in the original stool suspension is confirmed by comparison of theRNA electropherotypes induced in polyacrylamide gel. The serotype ofeach cell culture-adapted rotavirus may be determined by reaction withserotype-specific hyperimmune antisera to prototype rotaviruses preparedin rabbits and guinea pigs [Clark et al, (1987) supra].

EXAMPLE 2 Producing the Reassortants

A. WI79-3,9, WI79-4 and WI79-4,9

MA104 cell culture in a 24 well plate was washed twice with PBS andinoculated with 0.2 ml of a suspension containing 2.0×10⁵ pfu of humanserotype 1 strain WI79 rotavirus (WI79 was passaged eleven times inMA104 cells, including two plaque purifications, and thirteen times inCV-1 cells). This virus was allowed to absorb to cells for 60 minutes at37° C., after which the virus was removed and the cells washed twicewith PBS. 0.2 ml of a suspension containing 4.0×10¹ pfu of WC3 rotavirus(passage level 12) was added. The WC3 rotavirus was allowed to absorbfor 60 minutes, after which the cells were washed three times with PBSand fed with 1.5 ml of BHK medium with 13 micrograms/ml trypsin wasadded. Infected cells were incubated at 37° C. until CPE involved theentire monolayer (approximately 96 hours post infection).

The mixed infection was then harvested by three cycles of freezing andthawing. The cell culture fluids comprising this harvested infectionwere then reacted in a neutralization reaction consisting of addition ofcell culture fluids to an equal volume of hyperimmune rabbit antiserumto bovine serotype 6 rotavirus, obtained by conventional means anddiluted 1:50. The resulting neutralization mixture was then incubated at37° C. for 30 minutes, after which the surviving virus was plaqued onMA104 cell culture by standard technique. Plaques induced in MA104 cellculture were harvested at random, propagated in MA104 cell culture, andanalyzed by PAGE-SS for dsRNA electropherotype in comparison withparental rotaviruses WC3 and WI79.

Among these plaque isolates was a rotavirus reassortant which wasdeposited in two different passages on Nov. 25, 1987 with the ATCC anddesignated VR2194 and VR2196. This WC3/WI79 bovine/human rotavirusreassortant designated WI79-3,9 (originally WI79-9), contains gene 3 and9 from the human G1 serotype rotavirus WI79. Originally this reassortantwas designated WI79-9 to indicate that it contained the WI79 v.p.7encoding gene segment. The presence of human WI79 gene segment 3, whichruns closely between the human and bovine strains on the gels, wasconfirmed via polymerase chain reaction (PCR). However, gene 3 from WI79was always present in the reassortant as deposited.

WI79-3,9 was antigenically bivalent in virus neutralization (VN) testswith hyperimmune antisera. It reacts with antisera to bovine serotypeand human G1 rotaviruses. WI79-3,9 rotavirus reassortant replicates to atiter of 10⁷.0 pfu/ml in CV-1 cell culture. At this concentration, itwas completely attenuated for orally inoculated adults and infants asyoung as two months of age. In a high percentage of infants, WI79-3,9rotavirus reassortant induced virus neutralizing (VN) antibody specificfor rotavirus G1 and/or the bovine rotavirus serotype.

WC3/ WI79 bovine/human rotavirus reassortant WI79-4, containing gene 4from WI79 was also identified in this manner. WI79-4 was similarlyneutralized by antisera to human P1 rotavirus and by antisera to bovinerotavirus.

To generate WC3/ WI79 bovine/human rotavirus reassortant WI79-4,9, whichcontains gene segments 4 and 9 (v.p.4 and v.p.7) from the humanrotavirus strain WI79, reassortants WI79-3,9 and WI79-4 were combined ina culture and treated with anti-bovine rotavirus serum. WI79-4,9 wasthen identified among the progeny and harvested from the culture. Itcontained neither the v.p.4 or v.p.7 antigens from the bovine strain.WI79-4,9 was not neutralized at all by bovine antisera, even though itcontains nine bovine genes. It was neutralized by antisera to human typeG1.

B. WI61-4 and WI61-7,9

MA104 cell culture in a 24 well plate was washed twice with PBS andinoculated with 0.2 ml of a suspension containing 2.0×10⁵ pfu of humanstrain WI61 rotavirus (WI61 was passaged eleven times in MA104 cells,including two plaque purifications, and thirteen times in CV-1 cells).This virus was allowed to absorb to cells for 60 minutes at 37° C.,after which the virus was removed and the cells washed twice with PBS.0.2 ml of a suspension containing 4.0×10¹ pfu of WC3 rotavirus (passagelevel 12) was added. The WC3 rotavirus was allowed to absorb for 60minutes, after which the cells were washed three times with PBS and 1.5ml of BHK medium with 13 micrograms/ml trypsin was added. Infected cellswere incubated at 37° C. until CPE involved the entire monolayer(approximately 96 hours post infection).

The mixed infection was then harvested by three cycles of freezing andthawing. The cell culture fluids comprising this harvested infectionwere then reacted in a neutralization reaction consisting of addition ofcell culture fluids to an equal volume of hyperimmune rabbit antiserumto bovine serotype rotavirus, obtained by conventional means and diluted1:50. The resulting neutralization mixture was then incubated at 37° C.for 30 minutes, after which the surviving virus was plaqued on MA104cell culture by standard technique. Plaques induced in MA104 cellculture were harvested at random, propagated in MA104 cell culture, andanalyzed by PAGE-SS for dsRNA electropherotype in comparison withparental rotaviruses WC3 and WI61.

Among these plaque isolates was a WC3/WI61 bovine/human rotavirusreassortant designated WI61-4, containing gene 4 (v.p. 4) from humanstrain WI61. Also identified in this manner was WC3/WI61 bovine/humanrotavirus reassortant WI61-7,9 containing genes 7 and 9 (v.p. 7) fromWI61.

C. WI79-3+WISC2-9

Reassortant WI79-3+WISC2-9, which contains human gene 3 from WI79 andhuman gene 9 (encoding the human v.p.7 antigen) from WISC2 was preparedusing the methods described above. This reassortant was the result of amixed infection of WI79-3,9 and WISC2. The reassortant was selected forthe presence of human gene segment 9 from WISC2 via electrophoretic gel.The presence of human gene segment 3 from WI79 was later confirmed viaPCR analysis.

EXAMPLE 3 Method for Making WI79-3.9 Vaccine

Rotavirus reassortant WI79-3,9 was passaged a total of six times inMA104 cell culture which included three serial plaque purifications andthen was adapted to growth in CV-1 culture by three passages in CV-1cells. The third CV-1 cell passage represents a test vaccine evaluatedin adult volunteers and infants. For the vaccine trial provided inExample 6, this material was used as a seed pool for a vaccine lotpropagated in CV-1 cells, manufactured by Program Resources, Inc.(Rockville, Md.). Vero cells may also be used.

The test vaccine was produced in a manner similar to that used forWI78-1,6-11 in part A, above. Roller bottles (850 cm²) of CV-1 cellswere infected with WI79-3,9 rotavirus reassortant at a M.O.I. ofapproximately 0.30. Virus was adsorbed for thirty minutes at 37° C.,after which the cell cultures were fed with 100 ml/roller bottle of BHKmedium, serum-free, containing 25 micrograms/ml gentamicin and 1.0microgram/ml purified trypsin (Sigma Chemical Company). Infected cellcultures were incubated at 37° C. and harvested when CPE involved theentire monolayer at 72 hours post-infection.

Sterility tests consisted of inoculation of the vaccine into standardlaboratory media for the culture of aerobic and anaerobic bacteria,mycobacteria, and fungi. The vaccine was tested for mycoplasma byinoculation of 3T3 mouse cells in culture, followed by staining withHoechst stain for intracytoplasmic DNA. Testing for adventitious virusesincluded inoculation of human and primate cell cultures in the presenceof serotype-specific anti-rotavirus serum obtained by conventionalmethods, to suppress the replication of vaccine virus, which wereobserved for the appearance of CPE and/or hemadsorption. Adult andnewborn mice were inoculated intracerebrally and orally with the vaccineand observed subsequently for 30 days. Adult guinea pigs were inoculatedintraperitoneally and observed for 15 days post-inoculation.

Infectious rotavirus reassortant WI79-3,9 vaccine had an infectivitytiter of 10⁷.5 pfu/ml. The vaccine stock has been deposited with theAmerican Type Culture Collection, as ATCC No. VR2196.

EXAMPLE 4 Administration of WI79-3,9 Vaccines

Administration of vaccine to adults: Four adult volunteers were given afull dose (10⁷.5 pfu) of WI79-3,9 vaccine orally after oraladministration of 30 ml of MAALOX® to buffer stomach acids. All adultsremained clinically normal. None shed vaccine rotavirus in stool samplescollected three days post infection.

Administration of vaccine to infants: WI79-3,9 vaccine was administeredorally to infants in a volume of 2.5 ml, including 2.0 ml of vaccine and0.5 ml of cherry syrup. Infants were given 30 ml of infant formula, oroccasionally 1 ml/kg body weight of MAALOX® 30 minutes prior to vaccineto buffer stomach acids. In sequence, two infants were given a WI79-3,9dose of 10⁵.5 pfu; two were given a dose of 10⁶.5 pfu; and 49 infantsand one three year old child were given a dose of 10⁷.5 pfu. No vaccineassociated symptoms of disease were observed. Four of 50 infants given afull dose shed detectable levels of vaccine virus in stool. 30 of 54infants, or 57%, given any dose of vaccine developed avirus-neutralizing serum antibody response to one or more of rotavirusserotypes G1, G3, or bovine. This immune response to a primary dose ofWI79-3,9 was most often directed against the bovine serotype ofrotavirus, WC3, or serotype G1, WI79, reflecting the dual antigenicconstitution of the rotavirus reassortant.

The efficiency of induction of an immune response to WI79-3,9 in infantscould be further enhanced by giving a second "booster" dose of vaccineorally, 30 days after the primary dose. Such a booster could consist ofthe WI79-3,9 reassortant virus used for the original inoculation or avaccine consisting of either virus parent to the WI79-3,9 reassortant.The combined results with the WI79-3,9 virus vaccine followed by any ofthe three booster doses gave a 71% incidence of serum antibody responsein 2 to 4 month old infants and 91% in 5 to 11 month old infants.Following a booster dose, heterotypic antibody to serotype G3 (SA11)rotavirus was also induced with a frequency similar to that obtained tobovine serotype or with serotype G1 rotavirus. Thus, antibody wasinduced to the two serotypes, G1 and G3, most often responsible forrotavirus disease in infants in the United States.

Additional studies of this vaccine are described in detail in H F. Clarket al, J. Infect. Dis., 161:1099-1104 (1990) and H F. Clark et al,Vaccine, 8:327-333 (1990). Both references are incorporated herein byreference.

EXAMPLE 5 WI79-3,9 Vaccine Trial

A total of 325 infants were enrolled in the study, 237 in Rochester, and88 in Philadelphia. Randomization was conducted at a 2:1 ratio ofvaccine to placebo in Rochester, and at a 1:1 ratio in Philadelphia, sothat 207 infants received vaccine and 118 received placebo. There were13 infants who received one or more doses but did not complete thestudy, including 10 in the vaccine group, and 3 in the placebo group.Withdrawal from the study was because of relocation out of the area orreluctance to make study follow-up visits. No infants were withdrawnbecause of adverse events. These infants are included in the analysis ofreactions for doses that they received, but only the 312 infants whowere followed during the subsequent rotavirus season are considered inthe analysis of protective efficacy.

Vaccination took place primarily during the summer and fall. The firstdose of vaccine was administered on Jun. 18, 1992, and the last boosterdose was administered on Feb. 11, 1992. 257 subjects (79%) had receivedall 3 doses Jan. 1, 1993. Demographic features of the study populationwere similar in both vaccine and placebo groups, as shown in Table 3below.

                  TABLE 3                                                         ______________________________________                                        Characteristics of the Study Population                                                      Vaccine                                                                              Placebo                                                                (n = 207)                                                                            (n = 118)                                               ______________________________________                                        Mean age at      126.0    121.4                                               enrollment (days)                                                             Age range (days) 53-261   57-271                                              No. breast       74 (35.7)                                                                              55 (46.6)                                           feeding (%)                                                                   No. with sibs    72 (34.8)                                                                              36 (30.5)                                           ≦3 y.o. (%)                                                            No. in day       27 (13.0)                                                                              16 (13.6)                                           care (%)                                                                      ______________________________________                                    

A. Vaccine

The vaccine consisted of 10⁷.3 plaque forming units per dose of WI79-3,9virus administered by mouth in a total volume of 2.5 ml containing 0.25ml WI79-3,9 virus suspension, 1.75 ml sterile Eagle's minimal essentialmedia (MEM), and 0.5 ml cherry syrup (Philadelphia Extract Co.). TheWI79-3,9 virus was prepared as described in Example 2B above. Placeboconsisted of 2 ml of MEM and 0.5 ml cherry syrup. Vaccine and placebowere supplied as individual doses which were stored at minus 20° C. andthawed immediately prior to use.

B. Laboratory Studies

Diarrheal stools were tested for the presence of rotavirus by enzymeimmunoassay. Samples collected in Rochester were tested using thePathfinder (Kallestadt Diagnostics, Austin, Tex.), while those collectedin Philadelphia were tested using the Rotaclone (Cambridge Biosciences,Boston, Mass.) EIA kits. All positive samples were confirmed bypolyacrylamide gel electrophoresis [K. Dolan et al, J. Clin. Microbiol.,21:753-758 (1985)]. The serotype of the rotaviruses detected in thestudy was determined by polymerase chain reaction as previouslydescribed [J. Gentsch et al, cited above and V. Gouvea et al, J. Clin.Microbiol., 28:276-282 (1990)].

C. Clinical Studies

Infants in the study were healthy, between the ages of 2 and 8 months atthe time of the first vaccination, and had received at least oneprevious dose of oral polio and DPT vaccine. Infants were excluded fromparticipation if they had significant chronic illness. Vaccination waspostponed if symptoms of acute gastrointestinal illness or fever werenoted. The number and ages of household contacts, attendance at daycare, and breast feeding history were recorded at enrollment.

Infants were assigned to receive vaccine or placebo in double-blindfashion using a block randomization scheme. Three doses of vaccine orplacebo were administered, separated by approximately 2 month intervals.Subjects were not fed for one hour before each vaccination, and breastfeeding was withheld for 1 hour afterwards as well. Subjects enrolled inRochester received 30 ml of soy-based infant formula containing 400 mgsodium bicarbonate, followed by 2.5 ml of vaccine or placebo. At thePhiladelphia site, subjects were given 2.5 ml of vaccine or placeboafter ingesting at least 30 mls of either soy or milk-based infantformula. No sodium bicarbonate was added to the formula of thePhiladelphia subjects. The interval between study vaccination andreceipt of other routine vaccinations was at least 7 days.

Each family was given symptom cards, a thermometer, and an instructionsheet and materials for stool specimen collection after eachvaccination. Parents were requested to keep a record of the number andconsistency of each stool passed by subjects in the first 7 days aftereach vaccination, and to measure the rectal temperature of the infantdaily between 4 and 8 pm. In addition, a member of the study team calledthe parents once between days 3 and 5 following vaccination for directascertainment of reactogenicity.

Participants were monitored throughout the following winter and springto determine the incidence of rotavirus gastroenteritis. Surveillancecommenced on Jan. 1, 1993 with the first reported case of rotavirus inongoing community surveillance programs, and continued until Jun. 15,1993 which was 2 weeks after the last case of rotavirus gastroenteritisin the surveillance program at either site. Participating families werecontacted by telephone on a weekly basis during this period to inquireabout symptoms of gastroenteritis. Parents collected at least one stoolspecimen from the subject, and recorded daily temperatures and otherinformation during gastrointestinal illness, including the presence ofirritability, the number of episodes of vomiting, and the number andconsistency of stools.

The following definitions were used in the study: gastroenteritis wasdefined as the presence of one watery stool or three or more liquidstools and/or one or more episodes of vomiting in a 24 hour period;rotavirus gastroenteritis was defined as the presence of gastroenteritiswith identification of rotavirus in at least one stool specimen; andclinically significant rotavirus gastroenteritis was defined asrotavirus gastroenteritis with a clinical symptom score of >8, using ascoring system previously utilized in evaluating the efficacy of the WC3virus [H F. Clark et al, cited above]. Gastroenteritis episodes werescored according to the information provided by the parents during thesurveillance period. All diagnoses and gastroenteritis scores wereconfirmed by at least 2 members of the study team prior to unblinding ofthe study.

D. Results The statistical significance of differences in rates betweenvaccine and placebo groups was calculated using corrected chi-squaretests or Fisher's exact test as appropriate. Confidence limits forprotective efficacy were calculated as described by D. Kleinbaum et al,Lifetime Learning Publications (1982).

1. Reactions Following Vaccination

The WI79-3,9 virus was well tolerated at a dose of 10⁷.3 PFU in younginfants. Rates of reactions were generally similar in infants enrolledin Rochester and Philadelphia, and the data have been combined forpresentation in Table 4 below.

                                      TABLE 4                                     __________________________________________________________________________    Rates of Reactions within 7 days of Administration of WI79-3,9 Virus          Vaccine                                                                                       No. of subjects (%*) with following events                              No. (%)                                                                             with 7 days of indicated dose of vaccine                                Returning                                                                          Temp ≧      Altered                                     Group                                                                              Dose Records                                                                            38.0° C.                                                                      vomiting                                                                           diarrhea                                                                             behavior                                    __________________________________________________________________________    Vaccine                                                                            Dose 1                                                                             193 (93.2)                                                                         16 (8.3)                                                                             29 (15.0)                                                                          29 (15.0)                                                                            60 (31.1)                                   (n = 207)                                                                          Dose 2                                                                             183 (88.4)                                                                         22 (12.0)                                                                            18 (9.8)                                                                           51 (27.9)                                                                            45 (24.6)                                        Dose 3                                                                             175 (84.5)                                                                         37 (21.1)                                                                            18 (10.3)                                                                          41 (23.4)                                                                            42 (24.0)                                   Placebo                                                                            Dose 1                                                                             113 (95.8)                                                                         16 (14.1)                                                                            26 (23.0)                                                                          31 (27.4)                                                                            40 (35.4)                                        Dose 2                                                                             103 (87.3)                                                                         17 (16.3)                                                                            21 (20.4)                                                                          28 (27.2)                                                                            31 (30.1)                                        Dose 3                                                                              95 (80.5)                                                                         18 (18.9)                                                                            11 (11.6)                                                                          26 (27.3)                                                                            29 (30.5)                                   __________________________________________________________________________     * = Reported as percent of those returning records                       

There were no reactions that occurred at significantly higher frequencyin vaccine than in placebo recipients. In particular, the rate of feverfollowing vaccination was low, and was similar in vaccine and placeborecipients. When fever occurred it was generally low grade. Rectaltemperatures of ≧38.6° C. following dose 1, 2, or 3 were seen in 2.1,2.7, and 6.2% of vaccine recipients, and in 4.4, 2.9, and 4.2% ofplacebo recipients respectively. There was also no evidence ofclustering of days of fever in vaccine recipients, as approximatelyequal proportions of subjects in each group were febrile on each of theseven days following vaccination.

2. Protection From Rotavirus Gastroenteritis

The epidemic curves of the outbreaks of rotavirus gastroenteritis inRochester and Philadelphia were typical of rotavirus in these geographicareas. The first case of laboratory documented rotavirus gastroenteritiswas noted in week 2 of 1993 in Rochester and in week 5 of Philadelphia.Although some subjects experienced more than one episode ofgastroenteritis during the surveillance period, no subject experiencedmore than one episode of rotavirus gastroenteritis at either study site.

The distribution of episodes of gastroenteritis in vaccine and placeborecipients at both sites during the surveillance period is shown inTable 5 below. In this table, the subjects were followed during therotavirus season. For purposes of this study, rotavirus gastroenteritiswas defined as occurrence of vomiting and/or of one watery stool orthree or more loose stools in association with detection of rotavirus inan acute stool specimen.

                                      TABLE 5                                     __________________________________________________________________________    Protective Efficacy of the WI79-3,9 Vaccine Virus Against all Rotavirus       (RV)                                                                          Gastroenteritis and Clinically Significant RV Gastroenteritis in the          Surveillance Period                                                                                                 % Protection                                                 % Protection                                                                           No. Subjects (%)                                                                      Vs. Clinically                                        No. of Subjects                                                                      Against RV                                                                             with Clinically                                                                       Significant                                       No. of                                                                            (%) with RV                                                                          Gastroenteritis                                                                        Significant RV                                                                        Gastroenteritis                         Site  Group                                                                             Subjects                                                                          gastroenteritis                                                                      (95% Cl) Gastroenteritis                                                                       (95% Cl)                                __________________________________________________________________________    Rochester                                                                           placebo                                                                            74 19 (25.7)                                                                            64.1 (32.5, 80.9)‡                                                          12 (16.2)                                                                              83.7 (51.4, 94.6)§                      vaccine                                                                           152 14 (9.2)        4 (2.6)                                         Philadelphia                                                                        placebo                                                                            41  7 (17.1)                                                                            74.0 (-18.2, 94.3)|                                                            6 (14.6)                                                                             100¶                                vaccine                                                                            45 2 (4.5)         0 (0.0)                                         Total placebo                                                                           115 26 (22.6)                                                                            64.1 (35.9, 79.9)**                                                                    18 (15.7)                                                                              87.0 (62.6, 95.5)††            vaccine                                                                           197 16 (8.1)        4 (2.0)                                         __________________________________________________________________________     ‡--P = 0.001,                                                      §--P = 0.0001,                                                           |--P = 0.078,                                                        ¶--confidence limits not calculated, P = 0.009,                     **--P = 0.0004,                                                               ††--P = 0.000008.                                          

The WI79-3,9 virus vaccine was highly effective in the prevention ofclinically significant rotavirus gastroenteritis, as defined by aseverity score of >8. The differences between vaccine and placeborecipients in the proportions of subjects experiencing clinicallysignificant rotavirus gastroenteritis were statistically significant atboth study sites, with an 84% reduction in Rochester (P=0.0001) and a100% reduction in Philadelphia (P=0.009). When the results at the twostudy sites were combined, the vaccine protective efficacy againstclinically significant rotavirus gastroenteritis was 87.0%. Clinicallysignificant rotavirus disease occurred in only 4 of 195 (2.1%) infantsreceiving vaccine. Vaccines was also associated with a 64.1% reductionin all rotavirus episodes in Rochester (P=0.001) and a 74.0% reductionin Philadelphia (P=NS).

As noted previously, 21% of the subjects did not receive all three doseof vaccine before the beginning of the circulation of wild-typerotavirus in the community. Therefore, asymptomatic wild-type infectionscould have contributed to the protective efficacy seen in the study.However, the protective efficacy of the WI79-3,9 vaccine was similarwhen only those children who completed all three doses of vaccine beforeJanuary 1 were considered. Rotavirus gastroenteritis was seen amongchildren who completed all three doses before this date in 20 of 96placebo recipients and 15 of 161 vaccine recipients (55.3% protectiveefficacy, P=0.009), and clinically significant rotavirus gastroenteritiswas seen in 14/96 such placebo recipients and 3/161 such vaccinerecipients (87.2% protective efficacy, P=0.00007).

Since rotavirus is known to cause a relatively severe dehydratinggastroenteritis, it was also of interest to determine the overall effectof vaccination with WI79-3,9 vaccine on gastroenteritis-relatedmorbidity in the study population. There were no hospitalizations oremergency room visits for gastroenteritis in the study participants.However, subjects who received the WI79-3,9 vaccine had lower rates ofseveral measurements of gastroenteritis morbidity (Table 6).

                  TABLE 6                                                         ______________________________________                                        Impact of Winter Gastroenteritis in the Study Population                               No. (No. per subject)                                                                          Percent                                                      in those receiving:                                                                            Reduction                                                     Placebo      Vaccine    in Vaccine                                  Event     (115 subjects)                                                                             (197 subjects)                                                                           Recipients                                  ______________________________________                                        Episodes of any                                                                         79 (0.69)    81 (0.41)  40.1%                                       gastroenteritis                                                               Episodes of                                                                             34 (0.30)    20 (0.10)  65.7%                                       clinically                                                                    significant                                                                   gastroenteritis                                                               Days of   377 (3.28)   311 (1.58) 51.8%                                       gastrointestinal                                                              illness                                                                       MD visits 26 (0.23)    23 (0.12)  48.4%                                       Episodes  24 (0.21)    25 (0.13)  39.2%                                       requiring ORT                                                                 ______________________________________                                    

Overall, the risk of experiencing any clinically significantgastroenteritis was reduced 64.9% by vaccination. In addition, theaverage number of days of gastroenteritis per child during theobservation period was reduced by 50.6% in the vaccine recipients.

Vaccine recipients experienced 317 fewer days of diarrhea than expectedbased on the rate in the placebo group, and required 20 fewer thanexpected pediatrician visits for gastroenteritis. Other measures ofgastroenteritis morbidity, including use of oral rehydration,antipyretics, and episodes treated empirically with antimicrobialtherapy were similarly reduced in vaccine recipients.

3. Serotype of Natural Rotavirus Challenge

Serotype G1 clearly predominated in each community. All of therotaviruses detected in the Philadelphia area were serotype G1 by PCRtyping. Of the 19 symptomatic rotavirus infections in placebo recipientsin Rochester, 15 were G1, 1 was G4, and 3 samples did not havesufficient material for typing. Of the 14 symptomatic infections invaccine recipients, 11 were type G1, 1 was type G3, and 2 were unable tobe typed. PCR analysis for human P type was also performed on 16 samplesfrom Rochester (7 from vaccine and 9 from placebo recipients), and allwere P type 1.

E. Conclusions

This study demonstrates that the WI79-3,9 rotavirus, a reassortantbetween the bovine rotavirus WC3 and the human serotype G1 rotavirusWI79, is well tolerated when given as a three dose regimen in infants,and is highly effective at prevention of clinically significant serotypeG1 human rotavirus infection. Infants immunized with the WI79-3,9 virushad lower rates of rotavirus gastroenteritis, less diarrhea, andrequired less pediatric care than did placebo recipients. Confidencelimits (Cl) for efficacy were calculated as described by Kleinbaum etal, Epidemiologic Research: Principles and Ouantitative Methods,Lifetime Learning Publications (1982). The protective efficacy of theWI79-3,9 vaccine was 87.0% (95% Cl 62.6-95.5%) against clinicallysignificant rotavirus gastroenteritis (rotavirus gastroenteritis with aclinical severity score of >8), and was 64.1% (95% Cl 35.9-79.9%)against all rotavirus episodes. This level of protective efficacy of theWI79-3,9 virus is similar to that previously reported for two doses ofthe WI79-3,9 virus in small field trials conducted in Philadelphia [H F.Clark et al, cited above] and Rochester [C. Christy et al, cited above]during predominantly serotype 1 rotavirus seasons. Thus, the WI79-3,9virus has several desirable qualities as a live vaccine, i.e., it ishighly attenuated in the target population, and induces effectiveprotective immunity. Since the virus appears to be shed at extremely lowlevels in the stools of infected individuals, it is also unlikely to betransmitted efficiently to susceptible contacts, and unlikely to undergogenetic reversion during replication in vaccinees, although thesecharacteristics have not been studied extensively.

In this trial, post season sera were not obtained, so the rate ofrotavirus infection was not evaluated serologically. It is unlikelyhowever, that the WI79-3,9 vaccine prevented asymptomatic rotavirusinfection, based on the results of previous trials of the WI79-3,9 virus[H F. Clark et al, cited above] and other live, attenuated rotavirusvaccines [H F. Clark et al, cited above and H. Madore et al, J. Infect.Dis., 166:235-243 (1992)].

EXAMPLE 6 Immunogenicity of Heterologous v.p.4/v.p.7 RotavirusReassortant Vaccine

The following study illustrates that a WC3/human reassortant compositioncontaining human v.p.4 and human v.p.7 on separate reassortants elicitsa stronger immune response than a composition containing human v.p.4 andhuman v.p.7 in a single reassortant.

In infants, a clinical trial was conducted in which the administrationof the WI79-4,9 vaccine, containing a reassortant having both human v.p.4 and human v.p. 7 antigens therein, was compared with administration ofan equal-titered vaccine mixture of WI79-4 (human v.p. 4 and bovine v.p.7 antigens) and the human type 1 v.p.7 reassortant WI79-3,9 (bovine v.p.4 and human v.p. 7 antigens).

The infants, who were between 2 and 11 months of age, were first testedfor the presence of rotavirus neutralizing antibody in a conventionalserum virus neutralization antibody assay. For these tests, blood waswithdrawn, the serum separated, and the assay was performed using eachparent rotavirus, e.g., WC3 and WI79. Infants were scored eitheroriginally sero-negative or originally sero-positive.

Then a group of infants was administered orally a 2.5 ml dose of theWI79-4 and WI79-3,9 vaccine mixture or the WI79-4,9 vaccine. Thirty dayslater, the infants were administered orally a second 2.5 ml dose of thesame vaccine they received before. At the administration of the seconddose and again 30 days after the second dose of vaccine, the infantswere tested for the presence of rotavirus neutralizing antibody in thesame assay.

According to this trial, a positive immune response to either parentalvirus (WC3 or WI79) in the serum virus neutralization assay was definedas the development of a serum virus neutralization titer greater than1:125 in an originally sero-negative infant. In the case of anoriginally sero-positive infant, an increase in the titer of three-foldor more was considered a positive immune response.

The results obtained are reported in terms of virus neutralizingantibody. Results reveal that two doses of mixed WI79-4 and WI79-3,9vaccine induced neutralizing antibody to WC3 in 27/27 infants and totype 1 WI79 rotavirus in 21/27 (78%). Two doses of the reassortantWI79-4,9 vaccine induced antibody to type 1 WI79 rotavirus in 9/26infants (35%) and to WC3 in 7/27 infants (26%).

These results illustrate that the mixture of reassortants each havingheterologous species as well as human type rotavirus surface antigens(i.e., human and bovine v.p.4 and v.p.7 containing reassortants) is moreimmunogenic in infants than a reassortant bearing exclusively human typevirus surface antigens (i.e., human v.p.4 and human v.p. 7).

EXAMPLE 7 Ouadrivalent Reassortant Vaccine

The quadrivalent vaccine contained WI79-3,9 (G1), WISC2-4,9 (G2), WI78-8(G3), and WI79-4 (P1). Vaccine formulation was essentially as describedfor the WI79-3,9 trial in Example 5. Preliminary analysis of this trialis as follows.

Briefly, vaccine protocol was a pilot, double-blind, placebo-controlled,multicenter trial to evaluate the efficacy in healthy infants of a 3dose regimen. Each infant was enrolled to receive the first dose at 2-6months of age, with the second and third doses approximately 2 and 4months after dose 1. For evaluation of efficacy, a case of rotavirusdisease in study participants had to meet both of the followingcriteria: (1) three or more watery or looser than normal stools with a24 hour period and/or forceful vomiting. (2) Rotavirus identified byELISA in a stool specimen taken within 7 days of the onset of symptoms.Electropherotype analysis for virus genomic RNA was employed in order toconfirm the presence of rotavirus in fecal specimens. For finalanalysis, 199 evaluable subjects received vaccine and 206 receivedplacebo.

Based on ELISA positive cases, the rotavirus vaccine was 67.1%efficacious (95% Cl of [39.9%, 81.9%]) in preventing all disease and68.6% efficacious (95% Cl of [36.4, 84.5%]) in preventing clinicallysignificant disease. Statistical analysis revealed that the proportionof rotavirus cases in the vaccine group (14/199) was significantly lower(p<0.001) than the proportion of rotavirus cases in the placebo group(44/206). The proportion of clinically significant cases in the vaccinegroup (10/199) was also significantly lower than the proportion ofclinically significant cases in the placebo group (33/206).

Based on ELISA and electropherotype positive cases, the rotavirusvaccine was 72.9% efficacious (95% Cl of [47.3%, 86.0%]) in preventingall disease, and 72.6% efficacious (95% Cl of [42.9%, 86.9%]) inpreventing clinically significant disease. Statistical analysis revealedthat the proportion of rotavirus cases in the vaccine group (11/199) wassignificantly lower (p<0.001) than the proportion of rotavirus cases inthe placebo group (42/206). The proportion of clinically significantcases in the vaccine group (9/199) was also significantly lower than theproportion of clinically significant cases in the placebo group(34/206).

Severity scores >16 were noted in 11/42 of placebo recipients (26%)while only one vaccine recipient had a severity score of 16, and novaccine recipient had a score >16, indicating that the vaccine washighly effective in preventing the most severe cases of rotavirusdisease.

In order to detect vaccine shedding, 854 of 859 available fecalspecimens have been cultured. No shedding was detected in placeborecipients. Rotavirus shedding was observed in 13 specimens from 12vaccine recipients. Nine of these episodes occurred 3-5 days after thefirst vaccine dose and two participants shed rotavirus at day 7-9 afterdose one. Electropherotype analysis of plaque-purified rotavirusindicated that the 12 vaccine recipients each shed P1 vaccine strain. In5 of these 12 cases, a second rotavirus was detected, which appears tobe serotype P1G1. The P1G1 serotype was most likely generated in vivorather than during subsequent tissue culture passage.

Numerous modifications may be made by one skilled in the art to themethods and compositions of the present invention in view of thedisclosure herein. Such modifications are believed to be encompassed inthe appended claims.

What is claimed is:
 1. A vaccine useful in reducing the clinicalsymptoms associated with gastroenteritis caused by infection withrotaviruses of multiple human serotypes, said vaccine comprisingmultiple rotavirus reassortants, each said reassortant containing atleast one bovine rotavirus gene segment and at least one human rotavirusgene segment, with the remaining reassortant gene segments derived fromsaid bovine rotavirus, said human rotavirus or both, and wherein atleast one said reassortant comprises the gene segment encoding thevirion surface protein (v.p.) 7 neutralization antigen derived from ahuman rotavirus strain selected from rotavirus serotypes G5, G8, G9 andG10.
 2. The vaccine according to claim 1 comprising at least tworeassortants comprising said v.p.7 antigen derived from said bovinerotavirus, said human rotavirus or both, and wherein each of said tworeassortants comprises the gene segment encoding the virion surfaceprotein v.p.7 neutralization antigen derived from a human rotavirusstrain selected from rotavirus serotypes G5, G8, G9 and G10, whereineach of said two reassortants contains a different serotype selectedfrom said group.
 3. The vaccine according to claim 1 comprising at leastthree reassortants comprising said v.p.7 antigen derived from saidbovine rotavirus, said human rotavirus or both, and wherein each of saidthree reassortants comprises the gene segment encoding the virionsurface protein v.p.7 neutralization antigen derived from a humanrotavirus strain selected from rotavirus serotypes G5, G8, G9 and G10,wherein each of said three reassortants contains a different serotypeselected from said group.
 4. The vaccine according to claim 1 comprisingat least four reassortants comprising said v.p.7 antigen derived fromsaid bovine rotavirus, said human rotavirus or both, and wherein each ofsaid four reassortants comprises the gene segment encoding the virionsurface protein v.p.7 neutralization antigen derived from a humanrotavirus strain selected from rotavirus serotypes G5, G8, G9 and G10,wherein each of said four reassortants contains a different serotypeselected from said group.
 5. The vaccine according to claim 1 whereinsaid human rotavirus strain is a serotype found in animal and humanrotavirus infections.
 6. The vaccine according to claim 5 wherein saidrotavirus serotype is known to infect humans and porcines.
 7. Thevaccine according to claim 1 wherein said vaccine contains additionalimmunizing antigens.
 8. The vaccine according to claim 1, wherein eachsaid reassortant contains one gene segment derived from said bovinerotavirus strain of serotype G6.
 9. The vaccine according to claim 1,wherein each said reassortant contains one gene segment derived fromsaid human rotavirus.
 10. The vaccine according to claim 1, whichfurther comprises a reassortant containing at least one bovine rotavirusgene segment and at least one human rotavirus gene segment, wherein saidreassortant comprises the gene segment encoding the virion surfaceprotein v.p. 7 neutralization antigen derived from a human rotavirusstrain selected from rotavirus serotypes G1, G2, G3 and G4.
 11. Thevaccine according to claim 1, which further comprises a rotavirusreassortant containing at least one bovine rotavirus gene segment and atleast one human rotavirus gene segment, wherein the gene segmentencoding the virion surface protein v.p. 4 neutralization antigen isderived from a human rotavirus strain.
 12. A vaccine useful in reducingthe clinical symptoms associated with gastroenteritis caused byrotavirus infection, said vaccine comprising multiple lyophilizedrotavirus reassortants, each said lyophilized reassortant containing atleast one bovine rotavirus gene segment and at least one human rotavirusgene segment, with the remaining reassortant gene segments derived fromsaid bovine rotavirus, said human rotavirus or both, and wherein atleast one said reassortant comprises the gene segment encoding thevirion surface protein (v.p.) 7 neutralization antigen derived from ahuman rotavirus strain selected from rotavirus serotypes G5, G8, G9 andG10.
 13. The vaccine according to 12, further comprising a lyophilizedreassortant containing at least one bovine rotavirus gene segment and atleast one human rotavirus gene segment, with the remaining reassortantgene segments derived from said bovine rotavirus, said human rotavirusor both, and wherein said reassortant contains the gene segment encodingthe virion surface protein v.p. 7 neutralization antigen selected fromthe group of human rotavirus strains consisting of serotypes G1, G2, G3,and G4.
 14. The vaccine according to 12 further comprising a lyophilizedrotavirus reassortant containing at least one bovine rotavirus genesegment and at least one human rotavirus gene segment, wherein the genesegment encoding the virion surface protein v.p. 4 neutralizationantigen is derived from a human rotavirus strain.
 15. A vaccine usefulin reducing the clinical symptoms associated with gastroenteritis causedby rotavirus infection, said vaccine comprising multiple rotavirusreassortants in a liquid formulation, each said reassortant containingat least one bovine rotavirus gene segment and at least one humanrotavirus gene segment, with the remaining reassortant gene segmentsderived from said bovine rotavirus, said human rotavirus or both, andwherein at least one said reassortant comprises the gene segmentencoding the virion surface protein (v.p.) 7 neutralization antigenderived from a human rotavirus strain selected from serotypes G5, G8, G9and G10.
 16. The vaccine according to 15 further comprising areassortant containing at least one bovine rotavirus gene segment and atleast one human rotavirus gene segment, with the remaining reassortantgene segments derived from said bovine rotavirus, said human rotavirusor both, and wherein said reassortants contain the gene segment encodingthe virion surface protein v.p. 7 neutralization antigen selected fromthe group of rotavirus serotypes G1, G2, G3, and G4.
 17. The vaccineaccording to 15 further comprising a rotavirus reassortant containing atleast one bovine rotavirus gene segment and at least one human rotavirusgene segment, wherein the gene segment encoding the virion surfaceprotein v.p. 4 neutralization antigen is derived from a human rotavirusstrain.
 18. A vaccine formulation adapted for parenteral administrationcomprising multiple rotavirus reassortants, each said reassortantcontaining at least one bovine rotavirus gene segment and at least onehuman rotavirus gene segment, with the remaining reassortant genesegments derived from said bovine rotavirus, said human rotavirus orboth, and wherein at least one said reassortant comprises the genesegment encoding the virion surface protein (v.p.) 7 neutralizationantigen derived from a human rotavirus strain selected from rotavirusserotypes G5, G8, G9 and G10, and an adjuvant.
 19. The vaccineformulation according to claim 18 further comprising a reassortantcontaining at least one bovine rotavirus gene segment and at least onehuman rotavirus gene segment, with the remaining reassortant genesegments derived from said bovine rotavirus, said human rotavirus orboth, and wherein said reassortants contain the gene segment encodingthe virion surface protein (v.p.) 7 neutralization antigen derived froma human rotavirus strain selected from rotavirus serotypes G1, G2, G3,and G4.
 20. The vaccine formulation according to claim 18 furthercomprising a rotavirus reassortant containing at least one bovinerotavirus gene segment and at least one human rotavirus gene segment,wherein the gene segment encoding the virion surface protein v.p. 4neutralization antigen is derived from a human rotavirus strain.
 21. Theformulation according to claim 18 wherein said vaccine containsadditional immunizing antigens suitable for parenteral administration.22. The formulation according to claim 18 wherein said adjuvant isaluminum hydroxide.
 23. A method of reducing the clinical symptomsassociated with gastroenteritis caused by rotavirus infection comprisingadministering to a patient a suitable dosage of a vaccine of claim 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or
 11. 24. The method according to claim 23,wherein said method of administration is parenteral.
 25. The methodaccording to claim 23, wherein said method of administration isintranasal.
 26. The method according to claim 23, wherein said method ofadministration is oral.
 27. The method according to claim 23, furthercomprising administering multiple dosages of said vaccine.
 28. Themethod according to claim 27, wherein said multiple dosages are threedosages.
 29. A method of reducing the clinical symptoms associated withgastroenteritis caused by rotavirus infection comprising administeringto a patient a suitable dosage of a vaccine of claim 12, 13 or
 14. 30.The method according to claim 29, wherein said method of administrationis parenteral.
 31. The method according to claim 29, wherein said methodof administration is intranasal.
 32. The method according to claim 29,wherein said method of administration is oral.
 33. The method accordingto claim 29, further comprising administering multiple dosages of saidvaccine.
 34. The method according to claim 32, wherein said multipledosages are three dosages.
 35. A method of reducing the clinicalsymptoms associated with gastroenteritis caused by rotavirus infectioncomprising administering to a patient a suitable dosage of a vaccine ofclaim 15, 16,
 17. 36. The method according to claim 35 wherein saidmethod of administration is oral.
 37. The method according to claim 35wherein said method of administration is parenteral.
 38. The methodaccording to claim 35 wherein said method of administration isintranasal.
 39. The method according to claim 35, further comprisingadministering multiple dosages of said vaccine.
 40. The method accordingto claim 39, wherein said multiple dosages are three dosages.
 41. Amethod of reducing the clinical symptoms associated with gastroenteritiscaused by rotavirus infection comprising administering to a patient asuitable dosage of a vaccine of claim 18, 19, 20, 21 or
 22. 42. Themethod according to claim 41, further comprising administering multipledosages of said vaccine.
 43. The method according to claim 42, whereinsaid multiple dosages are three dosages.